Fragile X-associated tremor/Ataxia syndrome (FXTAS) is one of a large class of human neurological disorders that result from instability and expansion of nucleotide repeats. In FXTAS, a CGG nucleotide repeat expands in the 5? untranslated region of the fragile X gene, FMR1, and triggers formation of aggregated protein inclusions in the patient brains. Our group found that the FXTAS CGG repeat gets translated into toxic homopolymeric proteins despite its location outside of a canonical open reading frame through a process known as repeat associated Non-AUG initiated (RAN) translation. RAN translated proteins accumulate in patient tissues and contribute to CGG repeat associated toxicity in multiple model systems. In this proposal, we will explore how this alternative translational initiation occurs mechanistically. Our preliminary data suggests that RAN translation at CGG repeats is selectively enhanced by cellular stress, which typically blocks protein synthesis. In parallel, CGG repeats directly elicit cellular stress and trigger stress granule formation. Our central hypothesis is that CGG RAN translation and cellular stress participate in a feed-forward loop that drives neurodegeneration. Our collaborative team will directly test this hypothesis by using biochemical techniques as well as drosophila, mouse and human induced pluripotent stem cell models of FXTAS. In Aim 1 we will determine how cellular stress selectively activates RAN translation, focusing specifically on initiation factors that underlie this process. In Aim 2, we will elucidate how CGG repeats elicit cellular stress and influence stress granule dynamics and whether interventions in this process impact repeat toxicity. In Aim 3, we will test whether selective blockade of cellular stress pathways can disrupt this feed forward loop and alleviate CGG repeat associated toxicity across disease models. Together, these studies will illuminate critical events in the pathogenesis of FXTAS and other nucleotide repeat expansion disorders while rigorously testing two complementary & innovative approaches to selective RAN translation blockade.